Dirofilaria immitis is the causative agent of the heartworm disease in dogs, cats and wild canids, and is occasionally transmitted to humans resulting in pulmonary dirofilariasis. Prevalence values as high as 40% are found in some areas of the United States (Falls and Platt, American Journal of Veterinary Research, 43:738-739 (1982), and routine diagnostic screening has been recommended in an effort to control and manage the disease (American Heartworm Society, Proceedings of the Heartworm Symposium, 1992 (ed. Soll, M. D.), pages 289-294).
Traditionally, diagnosis relied exclusively on the detection of microfilariae in the blood. However, this method proved inadequate since a significant percentage of animals harbor occult infection, in which adult worms are present but there are not circulating microfilariae. More recently, commercial antigen test kits have become available which detect circulating female worm antigens. While these tests are highly specific, they have been shown to lack sensitivity for pre-patent or non-patent infections (Courtney, et al., Proceedings of the Heartworm Symposium, 1986 (ed. Otto, G. F.), pages 77-82; Dzimianski and McCall, Proceedings of the Heartworm Symposium, 1986 (ed. Otto, G. F.), pages 83-86; Wong and Fuller, Proceedings of the Heartworm Symposium, 1986 (ed. Otto, G. F.), pages 99-105; Courtney Journal of the American Animal Hospital Association, 24:27-32 (1988); Wong and Thomford, Journal of the American Animal Hospital Association, 27:33-38 (1991)).
Similar problems are encountered in diagnosis and management of the related filarial parasites of humans which are responsible for lymphatic filariasis (Brugia malayi, B. timori and Wuchereria bancrofti) and onchocerciasis (Onchocerca volvulus). However, a number of antigens from these parasites have now been identified which may be useful in diagnosis. Ov33-3 (Ov33) from O. volvulus, was one of the first filarial antigens reported to possess immunodiagnostic potential (Lucius, et al., Journal of Experimental Medicine, 167:1505-1510 (1988a)). The antigen was subsequently cloned by a number of investigators (Ov33.3 (Lucius, et al., Journal of Experimental Medicine, 168:1199-1204 (1988b)); Oc3.6 (Chandrashekar, et al., Journal of Clinical Investigation, 88:1460-1466 (1991)); OvD5B (Celine Nkenfou, Thesis, xe2x80x9cMolecular Cloning of Genes Coding Antigens Specific For Onchocerca volvulus: Evaluation of Expressed Proteins For Use In The Diagnosis Of Onchocerciasisxe2x80x9d University of Cameroon (1993)) and additional studies using recombinant antigen corroborated the earlier findings (Chandrashekar, et al., supra, Lucius, et al., Tropical Medicine and Parasitology, 43:139-145 (1992); Nkenfou, supra.) Perhaps surprisingly, homologs of this antigen have been found in other filarial parasites including B. malayi (Bm33) (Dissanayake, et al., Molecular and Biochemical Parasitology, 62:143-146 (1993)) and Acanthocheilonema viteae (Av33) (Willenbucher, et al., Molecular and Biochemical Parasitology, 57:349-351 (1993)). Interestingly, Bm33 was also shown to possess diagnostic potential for lymphatic filariasis (Dissanayake, et al., supra).
More recently, we described a 33 kDa antigen (DiT33) from D. immitis which reacts with sera generated against recombinant OvD5B, indicating the presence of a homolog in heartworm also (Mejia, et al., Parasite Immunology, 16:297-303 (1994)). A similar molecule exists in the intestinal nematode Ascaris suum since Ov33, Av33 (Willenbucher, et al., supra) and Bm33 (Dissanayake, et al., supra) share significant homology to an aspartyl protease inhibitor of A. suum Aspi3 (Martzen, et al., Biochemistry, 29:7366-7372 (1990)). However, the A. suum protein has not been assessed for diagnostic potential.
The inability to detect pre-patent infection with D. immitis limits surveillance and control activities, and results in delays in the evaluation of new therapies. Therefore a procedure for detecting early infection would be a useful tool in the management of heartworm infection.
In accordance with the present invention, an antigen from D. immitis, DiT33, has been identified, purified and cloned. DiT33 as well as the antibody responses generated by DiT33 may be used in early (approximately 11 weeks post-infection) immunodiagnosis of heartworm infection in mammals, and in particular in cats and dogs. DiT33 prepared in accordance with the present invention is substantially free of other antigen determinants from D. immitis. 
More specifically, purification and expression of DiT33, which we have determined to be an Onchocerca volvulus Ov33 homolog was demonstrated in Dirofilaria immitis. Rabbit antiserum raised against a recombinant fusion protein of O. volvulus, MBP/OvD 5B (Ov33), was found to react with a 31-33 kDa glycoprotein (DiT33) of adult worms of D. immitis. An antibody response to MBP/OvD 5B was observed in dogs, as early as 11 weeks post infection with infective larvae of D. immitis, and in dogs with occult infection. Cats both experimentally and naturally infected with D. immitis also reacted strongly with the recombinant antigen. In contrast, sera from dogs receiving chemically-abbreviated infection or from animals harboring a variety of other helminths failed to react.
Isolation and characterization of a complete cDNA encoding DiT33 from D. immitis is also described. DiT33 is expressed in Escherichia coli as a fusion with matose-binding protein (MBP). Using this fusion system, serological analysis was performed using a panel of clinically defined dog sera, including samples from dogs infected with other common parasites which present a challenge for heartworm diagnosis. The results of this study confirmed that DiT33 is a member of the putative aspartyl protease (pepsin) inhibitor family, and indicate that DiT33 is a prime candidate as an early marker for D. immitis infection.